The present invention, in some embodiments thereof, relates to methods and recombinant lactic acid bacteria for the production of ethanol from biomass material by a consolidated bioprocessing approach.
Ethanol is an established alternative fuel from renewable resources. Today it is mainly produced from sugar or starchy biomass, limiting the environmental benefit and posing a competition for the raw materials with food industry. In the last decade research efforts have mounted to replace this 1st generation ethanol by the 2nd generation ethanol made from lignocellulosic feedstocks, including pretreatment, enzymatic hydrolysis, sugar fermentation and process design. Most of the processes developed toward industrial scale involve addition of enzymes for cellulose and hemicellulose hydrolysis and use of specific yeast strains engineered to utilize C5 and C6 sugars. Both achieving effective biomass hydrolysis and complete sugar conversion are essential for an economical process. Although enzyme producers have made substantial improvements in the recent years, cost of cellulase enzymes are still in the range of $0.5 to $1.0 per gallon of 2nd generation ethanol.
A process strategy that aims to circumvent this critical cost-increasing item is the consolidated bioprocessing approach. In CBP an organism or a mixed culture of organisms produces enzymes for hydrolysis of cellulose and hemicellulose in lignocellulosic biomass and ferments the C5 and C6 sugars into ethanol or other valuable products without addition of cellulolytic or hemicellulolytic enzymes. Several mesophilic and thermophilic cellulolytic as well as non-cellulolytic microorganisms with engineered cellulase activity are under development for the application in CBP [Olson D G, et al., Curr Opin Biotechnol 2011, 23:1-10; La Grange D C Appl Microbiol Biotechnol 2010, 87:1195-1208; Svetlitchnyi et al., Biotechnology for Biofuels 2013, 6:31].
Conversion of lignocellulose to ethanol requires ethanol-tolerant microorganisms capable of degrading lignocellulose to fermentable sugars and fermenting the various sugars (pentoses and hexoses) released due to the hydrolysis of the lignocellulosic biomass.
Lactobacillus plantarum is a common lactic acid bacterium used in a variety of industrial and agricultural applications. L. plantarum prospers in environments containing lignocellulosic plant biomass. For example, in agriculture, these organisms are employed for conservation of lignocellulosic plant biomass for use in animal feed. In a process called ensilage, they quickly dominate the microbial population and produce lactic and acetic acids, thereby causing a pH drop which suppresses other microbial and fungal species. This bacterium was reported to possess high tolerance to ethanol concentrations in the media (up to 13% (v/v)).
L. plantarum is also able to metabolize pentose and hexose sugars derived from the lignocellulosic biomass. These attributes provide the bacterium with distinct advantages over the commonly used ethanol-producing yeast, Saccharomyces cerevisiae, which, in its native form, does not metabolize pentose. Another advantage is its acid tolerance which enables production of ethanol at low pH, thus reducing possible contamination by other bacteria and fungi and sparing handling steps due to acidic conditions sometimes imposed by pretreatment procedures. Furthermore, recent developments in the molecular biology of these bacteria include novel protein expression systems and the availability of the L. plantarum full genome sequence. Convenient genetic manipulation and robust expression of foreign genes render the genetic manipulations of these bacteria an accomplishable task.
U.S. Patent Application No. 20100129885 teaches microorganisms including Lactobacillus plantarum which are genetically modified to express cellulases and enzymes which are part of the butanol biosynthetic pathway.
U.S. Patent Application No. 20120190090 teaches microorganisms genetically modified to express cellulases and enzymes which are part of the butanol biosynthetic pathway.
Solem et al [Appl. Environ. Microbiol. 2013, 79(8):2512] teaches genetic engineering of Lactococcus lactis for ethanol production.
Additional background art includes U.S. Patent Application No. 20110230682.